Abstract Mechanisms underlying mechanical properties of muscle-tendon units Atrophy, weakness, and injury of skeletal muscle are widely recognized as major contributors to age-related physical frailty, but the importance of changes with aging in extracellular matrix (ECM) of muscle and tendon are poorly understood. Preliminary data show dramatic changes in tendon mechanical properties with aging, with a marked increase in stiffness at the muscle end of the tendon but minimal change at the bone end. The extensive changes with aging in mechanical properties of TA tendons occur with no major change in total collagen content. The regional variation in tendon functional properties coupled with regional variation in underlying structure may contribute to the lack thus far of clear relationships between biochemical and biomechanical properties. Therefore, our goals are to clarify the mechanisms underlying (i) regional differences in mechanical properties along tendons and (ii) changes in mechanical properties with aging, and to determine the impact of tendon changes on muscle function and susceptibility to injury. The working hypotheses are that (i) regional differences in tendon mechanical properties are due to regional variation in collagen content and proteoglycan expression resulting in differences in ECM structure and (ii) with aging, the composition of the entire tendon becomes similar to that of the region nearest bone, causing the tissue to be stiffer and less extensible overall and increasing the likelihood of contraction-induced injury to muscle fibers. Experiments will be performed on tibialis anterior (TA) muscle-tendon units (MTU) and permeabilized single muscle fibers of adult (8 months), middle aged (24 months), and old (33 months) Fisher X Brown Norway rats. The Specific Aims are: 1. determine the impact of aging on passive mechanical properties of MTUs and the interaction between tendon stiffness and muscle fiber length during contraction, 2. elucidate mechanisms underlying the regional differences in mechanical properties along tendons and the changes in tendon mechanical properties that occur with aging, and 3. evaluate the effects of prior active shortening on force deficits caused by lengthening contractions (LCs) of permeabilized single fibers from muscles of adult and old rats. To address the Aims, our approach will be to (i) make high resolution optical measurements of stress-strain relationships along MTUs during stretches without activation and during muscle activation with and without stretch, (ii) perform thorough regional analyses of protein and gene expression, collagen content and cross-linking, and ECM structure of tendons to establish meaningful structure-function relationships, and (iii) apply, to maximally activated permeabilized single fibers from TA muscles of rats, pre-stretch shortening movements that span, in both amplitude and speed, the extension properties of tendons established in Aim 1 to determine the impact on muscle function and susceptibility to injury of alterations in the mechanical environment of fibers that are unavoidably created by changes in tendon properties. Narrative The relevance to public health is based on the impact of increasing susceptibility to muscle and tendon injury as contributors to the development with aging of physical frailty. Frailty causes immobility and falls and is a key factor limiting the ability to live independently. Our project will further understanding of the contribution of changes with aging in the mechanical properties of tendons and muscle fibers to the increased susceptibility to musculoskeletal injury. Clarifying mechanisms underlying changes with aging in the structure and function of muscle-tendon units will improve the likelihood of delaying the onset and progression of frailty.